Recent advances in understanding the metabolism of methylglyoxal in mammalian cells suggest that the glutathione (GSH)-dependent glyoxalase enzyme system is a useful target for antitumor drug development (Creighton et al, Drugs of the Future, 25:385-392 (2000)). The physiological function of this detoxification pathway is to remove cytotoxic methylglyoxal from cells as D-lactate via the sequential action of the isomerase glyoxalase I (GlxI) and the thioester hydrolase glyoxalase II (GlxII), as shown in Scheme 1 below (Creighton et al, “Glutathione-Dependent Aldehyde Oxidation Reactions”, In Molecular Structure and Energetics: Principles of Enzyme Activity, Liebman et al, Eds.; VCH Publishers, 9: 353-386 (1988)).

Methylglyoxal is a highly reactive alpha-ketoaldehyde that arises as a normal by-product of carbohydrate metabolism (Richard et al, Biochemistry, 30:4581-4585 (1991)) and is capable of covalently modifying proteins and nucleic acids critical to cell viability (Reiffen et al, J. Cancer Res. Clin. Oncol., 107:206-219 (1984); Ayoub et al, Leuk. Res., 17:397-401 (1993); Baskaran et al, Biochem. Int., 212:166-174 (1990); Ray et al, Int. J. Cancer, 47:603-609 (1991); White et al, Chem-Biol. Interact., 38:339-347 (1982); and Papoulis et al, Biochemistry, 34:648-655 (1995)).
Inhibitors of GlxI have long been sought as possible anticancer agents, because of their potential ability to induce elevated concentrations of methylglyoxal in tumor cells (Creighton et al (2000), supra), and because of the observation that rapidly dividing tumor cells are exceptionally sensitive to the cytotoxic effects of exogenous methylglyoxal (Ray et al, supra; White et al, supra; and Papoulis et al, supra). The basis of this sensitivity is not well understood, but appears to arise, in part, from methylglyoxal induced activation of the stress-activated protein kinases c-Jun NH2-terminal kinase 1 (JNK1) and p38 mitogen-activated protein kinase (MAPK), which leads to caspase activation and apoptosis (programmed cell death) in tumor cells (Sakamoto et al, Clinical Cancer Research, 7:2513-2518 (2001); and Sakamoto et al, J. Biol. Chem., 277:45770-45775 (2002)). Moreover, methylglyoxal is probably genotoxic, on the basis of its ability to covalently modify nucleotide bases in DNA (Papoulis et al, supra).
Further, inhibitors of hGlxI that are hydrolytically destroyed by the thioester hydrolase GlxII offer a selective strategy for specifically inhibiting tumor cells, as normal cells contain much higher concentrations of GlxII than tumor cells. Table 1 below shows a comparison of the activities of GlxI and GlxII in normal versus cancer cells (Creighton et al (2000), supra).
TABLE 1Reported Glyoxalase Activitiesin Normal Cells versus Cancer CellsGlyoxalase Activity(mU/mg protein)TissueGlxIGlxIIGlxI/GlxIINormalbrain (human)1113 ± 19817 ± 1561.4liver (human) 209 ± 56360 ± 130.6heart (hamster) 339 ± 24280 ± 471.2kidney (human) 323 ± 48330 ± 861.0lymphocytes (mouse) 360 ± 30200 ± 301.8Tumormelanoma B16 (mouse) 370 ± 160 66 ± 185.6leukemia L1210 (mouse) 310 ± 30 20 ± 315.5glioblastoma (human) 290 ± 56 53 ± 105.5fibroadenoma mammae 419 ± 73 27 ± 715.5(human)bladder HT-1107 542 ± 38 8 ± 167.8(human)prostate PC-3 (human)4206 ± 294 45 ± 393.4testis T1 (human)4767 ± 275 94 ± 1251.0colon HT29 (human) 542 ± 59 11 ± 149.3
Thus, normal cells should be able to detoxify thioester inhibitors of hGlxI much more rapidly than the corresponding tumor cells, resulting in much higher steady concentrations of the inhibitors in tumor cells. Consistent with this hypothesis, the diethylester prodrug form of S—(N-p-chlorophenyl-N-hydroxycarbamoyl) glutathione (CHG), which is both an inhibitor of hGlxI and a substrate for hGlxII (Murthy et al, J. Med. Chem., 37:2161 (1994)), is significantly more toxic to murine leukemia L1210 cells than to normal splenic lymphocytes in culture, reflecting the 10-fold lower activity of hGlxII in L1210 cells versus splenic lymphocytes (Kavarana et al, J. Med. Chem., 42:221-228 (1999)).
Since an ideal chemotherapeutic agent should be highly specific for cancer cells and therefore have minimal side effects, GlxI inhibitors are attractive anticancer agents because of their selective accumulation in tumor cells rather than normal cells.
Further, an antitumor strategy targeting hGlxI provides benefits over more established chemotherapies that attack rapidly dividing tumor cells at various stages of mitosis, or that arrest tumor cells at some stage in the cell cycle. For example, many of the small molecule antitumor drugs currently in use target rapidly dividing tumor cells by either directly or indirectly inhibiting DNA and/or protein synthesis. Thus, these drugs will also adversely affect rapidly dividing normal cells, like those of the intestinal epithelium and bone marrow. As a result, side-effects of antitumor agents currently in use often include myelosuppression, intestinal disorders, dose-dependent cardiotoxicity, pulmonary fibrosis, anaphylactic reactions, alopetia, and anorexia.
A class of transition state analogue inhibitors of hGlxI are known which are S—(N-aryl/alkyl-N-hydroxycarbamoyl)glutathiones. Specifically, these thioester derivatives of GSH mimic the stereoelectronic features of the tightly bound transition state species that flank the ene-diolate intermediate that forms along the reaction coordinates of the enzyme. As such, these compounds are the strongest known competitive inhibitors of hGlxI, with inhibition constants (Kis) in the mid-nanomolar range: S—(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione (CHG), Ki=46 nM; S—(N-p-bromophenyl-N-hydroxy-carbamoyl)glutathione (BHG), Ki=14 nM; S—(N-p-iodophenyl-N-hydroxy-carbamoyl)glutathione (IHG), Ki=10 nM; and S—(N-hexyl-N-hydroxycarbamoyl)glutathione, Ki=16 nM (Kalsi et al, J. Med. Chem., 43:3981-3986 (2000)). These transition state analogue inhibitors are hereinafter called “reversible competitive inhibitors.”
Moreover, the reversible competitive inhibitors are also slow substrates for bovine liver GlxII, which suggests that these compounds can selectively inhibit tumor cells over normal cells (Murthy et al, supra).
The effectiveness of the reversible competitive inhibitors can be measured by their specificity for the GlxI active site and by the time they occupy the active site, thereby blocking access of the enzyme's natural substrate (GSH-methylglyoxal thiohemiacetal). An inhibitor with a low competitive inhibition constant (Ki) associates with the active site of an enzyme with higher affinity and greater specificity, and therefore, occupies the active site for a longer period of time than inhibitors with higher Ki values.
The competitive inhibitors have been shown to be lethal to different human and murine tumor cell lines in culture when administered as diethyl ester prodrugs (U.S. Pat. No. 5,616,563) (Kavarana et al, supra). Di- or mono-ester prodrugs are employed since the charged glutathionyl precludes rapid diffusion into cells. After diffusion, these prodrugs undergo de-esterification inside the cell. The diethyl ester prodrugs of S—(N-phenyl-N-hydroxycarbamoyl)glutathione, PHG(Et)2; S—(N-p-bromophenyl-N-hydroxycarbamoyl)glutathione, BHG(Et)2; and S—(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione, CHG(Et)2, inhibit the growth of murine leukemia L1210 cells in culture with IC50 values of 63, 16, and 5 μM, respectively, after 72 hours of incubation.
In vivo efficacy studies with CHG(Et)2 by Sharkey et al, Cancer Chemother. and Pharmacol., 46:156-166 (2000), which is herein incorporated by reference, also support the hypothesis that competitive inhibitors of GlxI are potentially important antitumor agents. For example, bolus i.v. administration of CHG(Et)2 inhibited the growth of human prostate PC3 tumors in athymic nude mice. The potency of CGH (Et)2 was as good as the positive control cisplatin without the cisplatin-associated haemopoetic toxicity. However, no effect was observed by continuous infusion of CHG(Et)2 over 12 days, suggesting that, when administered by continuous infusion, not enough drug is available to be absorbed by such a rapidly growing tumor. Further, the efficacy of CHG(Et)2 against murine melanotic melanoma implanted subcutaneously in (Es-1e) mice was also evaluated. After, a preliminary pharmacokinetic study that showed significant accumulation of drug in melanoma following i.v. bolus administration of CHG(Et)2, the efficacies of CHG(Et)2 and the dicyclopentyl diester prodrug CHG(cyclopentyl)2 were evaluated. All of the drugs gave results that were statistically significant from the vehicle control. CHG(Et)2 and CHG(cyclopentyl)2 were as effective as the positive control adriamycin, but at much higher dosages. There were no detectable side-effects observed in any of the trials. Similar results were obtained with athymic nude mice bearing human colon HT29 tumors. Potency was as good as the positive control vincristin when the drug was administered by continuous i.v. infusion, without any detectable side-effects. However, efficacy was not observed by bolus administration, indicating that this slowly growing tumor was not sufficiently exposed to drug. Thus, the method of drug delivery (bolus infusion versus continuous infusion) is an important aspect of efficacy.
All three in vivo efficacy studies required high doses of prodrug to inhibit tumor growth (80 mg/kg body mass). A subsequent series of studies by Sakamoto et al (2001) showed that the dicyclopentyl diester of a weaker competitive inhibitor of GlxI, (S-p-bromobenzyl)glutathione (BBG), effectively inhibited lung tumor cell lines NCI-H460, NCI-H226, A549, NCI-H23, DMS273, DMS114, NCI-H522 in vitro, and DMS144 and human prostate DU145 in nude mice bearing human cancer xenografts. Further, it was discovered that BBG is effective against human lung tumors (DMS114) in mice at dosages of 100 mg/kg (Sakamoto et al (2001), supra), with no evidence of adverse side effects during the 21 days of administration. Taken together, these studies show that inhibition of GlxI is an important chemotherapeutic strategy in cancer control.
In order to improve this antitumor strategy, more effective GlxI inhibitors must be developed that will be effective at much lower dosages. The large dosages currently required for antitumor activity increase the probability that toxic side effects or adverse reactions will develop when these compounds are used over long periods of time. Thus, there is a need in the art for more potent inhibitors that have a greater binding affinity or specificity for the active site of GlxI such that they may be administered at lower dosages than known GlxI inhibitors to effectively inhibit tumor growth.